Fluctuations in circulating hormones, activation of the autonomic nervous system and altered mechanical work load are implicated as potential mediators of evolving hypertrophic heart disease. How the transduction of neurohumoral and load dependent signals modulate adult myocyte growth has been difficult to pursue in whole heart preparations; however, adult feline myocytes maintained in long term culture provide a unique approach to directly explore the cellular mechanisms that regulate myocyte growth. A fine balance must be struck between the rates of synthesis and degradation of cardiac proteins if the heart is to retain its normal size and function. Although much is known about the synthetic side of this equation, virtually nothing is known about rates of contractile protein degradation or the messengers and mechanisms that regulate their breakdown. The principal objective of this proposal is to determine the rates of total, contractile and cytoskeletal protein degradation in the cultured adult myocyte where experimental conditions can be precisely defined and controlled. The long-term stability of this preparation facilitates the study of protein breakdown because the half-lives of adult contractile proteins are measured in days rather than hours. To measure the degradation of long-lived and short-lived proteins, myocytes will be biosynthetically labeled with [14C] and [3H] amino acids and the radiolabeled heart cells then will be exposed to neurohumoral factors and/or mechanical stretch. Rates of degradation will be measured for total and purified proteins. Parallel immunofluorescence and immunoelectron microscopic approaches will be employed to correlate alterations in proteolysis with any changes in the reorganization of the contractile apparatus. Four proteolytic pathways will be investigated to determine whether they participate in the degradation of specific-classes of myocyte proteins. In addition, cAMP production, inositol phosphate turnover and [Ca2+]i will be monitored to assess whether these second messengers directly activate specific proteolytic pathways. Since adult heart cells synthesize proteins at rates identical to those documented in the whole animal, this stable myocyte culture model should divulge fundamental new observations on the mechanisms that regulate protein turnover in the adult heart. As such, the experiments outlined in this proposal will provide valuable insight into assessing whether any alteration in the rate of protein degradation may limit myocyte growth and, ultimately, pathophysiologic changes that develop during cardiac hypertrophy.